Sequence-specific binding of proteins to DNA, RNA, protein and other molecules is involved in a number of cellular processes such as, for example, transcription, replication, chromatin structure, recombination, DNA repair, RNA processing and translation. The binding specificity of cellular binding proteins that participate in protein-DNA, protein-RNA and protein-protein interactions contributes to development, differentiation and homeostasis.
Zinc finger proteins (ZFPs) are proteins that can bind to DNA in a sequence-specific manner. Zinc fingers were first identified in the transcription factor TFIIIA from the oocytes of the African clawed toad, Xenopus laevis. A single zinc finger domain of this class of ZFPs is about 30 amino acids in length, and several structural studies have demonstrated that it contains a beta turn (containing two conserved cysteine residues) and an alpha helix (containing two conserved histidine residues), which are held in a particular conformation through coordination of a zinc atom by the two cysteines and the two histidines. This class of ZFPs is also known as C2H2 ZFPs. Additional classes of ZFPs have also been suggested. See, e.g., Jiang et al. (1996) J. Biol. Chem. 271:10723-10730 for a discussion of Cys-Cys-His-Cys (C3H) ZFPs. To date, over 10,000 zinc finger sequences have been identified in several thousand known or putative transcription factors. Zinc finger domains are involved not only in DNA recognition, but also in RNA binding and in protein-protein binding. Current estimates are that this class of molecules will constitute about 2% of all human genes.
Most zinc finger proteins have conserved cysteine and histidine residues that tetrahedrally-coordinate the single zinc atom in each finger domain. In particular, most ZFPs are characterized by finger components of the general sequence:-Cys-(X)2-4-Cys-(X)12-His-(X)3-5-His-(SEQ ID NO:1), in which X represents any amino acid (the C2H2ZFPs). The zinc-coordinating sequences of this most widely represented class contain two cysteines and two histidines with particular spacings. The folded structure of each finger contains an antiparallel β-turn, a finger tip region and a short amphipathic α-helix. The metal coordinating ligands bind to the zinc ion and, in the case of zif268-type zinc fingers, the short amphipathic α-helix binds in the major groove of DNA. In addition, the structure of the zinc finger is stabilized by certain conserved hydrophobic amino acid residues (e.g., the residue directly preceding the first conserved Cys and the residue at position +4 of the helical segment of the finger) and by zinc coordination through the conserved cysteine and histidine residues.
Canonical (C2H2) zinc finger proteins having alterations in positions making direct base contacts, ‘supporting’ or ‘buttressing’ residues immediately adjacent to the base-contacting positions, and positions capable of contacting the phosphate backbone of the DNA have been described. See, e.g., U.S. Pat. Nos. 6,007,988; 6,013,453; 6,866,997; 6,746,838; 6,140,081; 6,610,512; 7,101,972; 6,453,242; 6,785,613; 7,013,219; PCT WO 98/53059; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416; Segal et al. (2000) Curr. Opin. Chem. Biol. 4:34-39.
In addition, zinc finger proteins containing zinc fingers with modified zinc coordinating residues have also been described (see, e.g., U.S. Patent Publication Nos. 20030108880, 20060246567 and 20060246588; the disclosures of which are incorporated by reference). However, while zinc finger proteins containing these non-canonical zinc fingers retain gene transcription regulatory function, their ability to act as zinc finger nucleases (ZFNs) is in some cases diminished relative to zinc finger proteins consisting exclusively of canonical, C2H2 zinc fingers.
Thus, there remains a need, particularly in the construction of zinc finger nucleases, for additional engineered zinc finger binding proteins containing zinc fingers having optimized non-canonical zinc coordinating regions.